Many phenolic compounds and aromatic amines are present in the effluents of chemical and dye manufacturers, coal processors, resin and plastic manufacturers, textile processors, and other industries. Since most phenols and aromatic amines are toxic and many are potentially carcinogenic, they must be removed from effluents in which they are present. Existing methods for the removal of phenols and aromatic amines from water include adsorption, extraction, microbial and chemical oxidation, electrochemical techniques, and irradiation. However, all of these methods suffer from serious shortcomings such as high cost, incompleteness of purification, formation of hazardous byproducts, and low efficiency. See, e.g., M. W. Slein et al, Degradation of Chemical Carcinogens, Van Nostrand Reinhold Co., 1980; Cleaning our Environment--A Chemical Perspective, American Chemical Society, 1978. Therefore, alternative methods of removing aromatic compounds from waste water are highly desirable.
Peroxidases are enzymes that catalyze chemical reactions that normally involve the transfer of hydrogen radicals from organic substances to substrates comprising peroxides. Such reactions may be complex, and may involve many different substances. However, the following simplified example explains the major functions performed by several substances in one such reaction. Horseradish peroxidase reacts with phenol by removing a hydrogen radical (one proton with one electron) from the hydroxide group on the phenol. The phenol is thereby converted to an aromatic free radical, which participates in a subsequent reaction that depends upon other substances that are present in the solution. The hydrogen radical reacts with hydrogen peroxide to form water.
The addition of both peroxide and peroxidase to an aqueous solution containing certain phenols or aromatic amines has long been recognized to cause color changes and precipitation of the aromatic compounds. Most of the work done to date involving peroxidase reactions with aromatic compounds has involved either: (1) the use of peroxidase as an indicator to determine whether phenols or aromatic amines are present in a solution: (2) research into the exact chemical reactions and products created. See, e.g., P. J. G. Mann et al, Proceedings of the Royal Society B119, p. 47 (1935); B. C. Saunders et al, Peroxidase, p. 10 et seq., Butterworth's (1964); A. Klibanov et al, "Horseradish peroxidase for the removal of carcinogenic aromatic amines from water," Enzyme Microb. Technol. Vol. 3, pp. 119-122 (1981); G. Galliani et al, "Horseradish Peroxidase-catalyzed Oxidation of Aromatic Tertiary Amines with Hydrogen Peroxide," J. Chem. Soc. Perkin I pp. 456-460 (1980). Each of these works deals with the reaction of a single aromatic substance in aqueous solution.
Aromatic free radicals are relatively reactive. The creation of aromatic free radicals in an aqueous solution tends to commence a chain reaction whereby aromatic molecules become bonded together to form an insoluble precipitate which can be removed from solution by sedimentation, filtration, centrifugation, or other conventional techniques. The efficiency of this technique varies between aromatic substances. For example, research by the Applicant indicates that removal efficiencies for various phenolic compounds under comparable conditions ranged from 53.5% efficiency for ortho-aminophenol and 85.3% for phenol, to 99.8% for ortho-chlorophenol. Removal efficiencies for various aromatic amines under comparable conditions ranged from 62.5% for 4-chloroaniline and 72.9% for aniline to 98.6% for meta-phenylenediamine. Such efficiencies are assessed by measuring the aromatic content of the water before the addition of peroxidase and peroxide, and after removal of the resulting precipitate, by spectrophotometric or other analytical methods.
The economic feasibility of peroxidase enzyme treatment to remove aromatic compounds from water depends upon the extent of polymerization and precipitation of the compounds. Since enzymes and peroxides tend to be relatively expensive, the usage of peroxidase and peroxide to treat large volumes of wastewater would not be feasible unless the compounds were removed to a very high degree. Since phenol, aniline and many other aromatic compounds are not removed to a high degree of efficiency by peroxidase treatment, it appeared to persons skilled in the art that peroxidase treatment was not an economically feasible method of treating most forms of wastewater.